Treatment of seed and plants with useful bacteria

ABSTRACT

An isolate of useful bacteria, preferably of the genus Pseudomonas, preferably of the species Proradix, is used for the treatment of plants and/or seed and incubated in a culture medium containing phosphorus compounds, nitrogen compounds and succinic acid. The solution can be used directly for spraying plants and/or seed, optionally followed by vacuum treatment. Furthermore, the solution can be vacuum-dried, the powder being dissolved in water prior to use.

RELATED APPLICATION

[0001] This application is a continuation of International PatentApplication PCT/EP00/10903, filed on Nov. 4, 2000 which designates theUnited States, published as WO 01/40441 in a language other thanEnglish.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the treatment of seed and plantswith bacteria of the genus Pseudomonas and to a new species of the genusPseudomonas, which is particularly suitable for this purpose.

[0004] 2. Related Prior Art

[0005] A method of strengthening and protecting plants in whichmicroorganisms of the genus Pseudomonas are employed is alreadydisclosed in DE 197 39 364 A1.

[0006] In the known method, at least one resistance inductor togetherwith a useful microorganism is introduced into the plants' nutrientmedium or into the seed of the plant, resistance inductor andmicroorganism having a complementary positive effect on the plant.

[0007] An example of a useful microorganism which may be mentioned isthe strain Pseudomonas sp. Psl of the genus Pseudomonas fluorescens,which is deposited at the DSMZ: (Deutsche Sammlung von Mikroorganismenund Zellkulturen [German Collection of Microorganisms and CellCultures]).

[0008] This publication furthermore describes a method of how to isolatesuch a useful microorganism from soil and roots. In this method, adilution series from soil and roots is plated out, incubated and testedfor fluorescent colonies which are then used in a screening againstharmful fungi. Furthermore, a strain found by the known method isstudied for its combination ability with resistance inductors. In thismanner, the strain Pseudomonas sp. Psl has been found.

[0009] However, experiments carried out by the applicant of the presentapplication have revealed that the treatment described in thepublication stated at the outset is as yet unsatisfactory, which can beattributed, inter alia, to the fact that expensive resistance inductorsmust be employed together with the microorganism described, and it hasfurthermore been revealed that the protective action is frequentlyunsatisfactory.

[0010] U.S. Pat. No. 2,932,128 discloses a method for the treatment ofseed in which the seed is moistened with a bacterial solution and thenexposed to a vacuum in order to impregnate the seed.

SUMMARY OF THE INVENTION

[0011] In view of the above, it is an object of the present invention toprovide novel solutions and methods and a novel microorganism for thetreatment of seed and/or plants.

[0012] In accordance with one object, the invention relates to aPseudomonas strain termed Proradix, which was deposited under theprovisions of the Budapest Treaty on Mar. 11, 1999 at the DSMZ, 38124Braunschweig under the deposit number Pseudomonas sp. PRORADIX-DSM13134.

[0013] The Pseudomonas strain Proradix, which, according to studies bythe DSMZ, probably constitutes another species within RNA group I of thePseudomonadaceae, has been isolated by the inventor of the presentapplication. A final attribution of Proradix was not possible, however,since only relatively little sequence similarity exists with validlydescribed species of the genus Pseudomonas.

[0014] Field trials with the species Proradix have shown that it is auseful bacterium which is capable of colonizing the roots of usefulplants, and there can exert its effect on the plant. It has beendemonstrated in that connection that one application per vegetationperiod suffices and that additional resistance inductors can bedispensed with.

[0015] Proradix can be employed in a formulation as pickling liquor oras a powder and is suitable for the treatment of vegetable seed, inparticular lamb's lettuce and carrots, of lawn seed and seed of woodyspecies. Furthermore, it may be employed in green-manure seed for thebiological soil conditioning and de-contamination, and for the seedtreatment and immersion treatment of potatoes. In all theseapplications, Proradix has shown an improved quality compared tountreated control groups and in some cases also in comparison withchemical fungicides.

[0016] According to another object, the invention relates to a methodfor the preparation of a solution for the treatment of plants and/orseed, comprising the steps:

[0017] a) providing an isolate of useful bacteria, preferably of thegenus Pseudomonas,

[0018] b) providing a culture medium containing phosphorus compounds,nitrogen compounds and succinic acid,

[0019] c) inoculating the culture medium with the isolate, and

[0020] d) incubating the culture medium at approximately 26-32° C. withgentle shaking for at least 50 hours.

[0021] The inventor has recognized that a solution thus prepared bringsabout a conditioning of the useful bacteria, which contributes to animproved protective action in the plants/seed treated with the bacteria.The inventor's current explanation of this conditioning effect is theinduction of the formation of exopolysaccharides due to degradationproducts of the succinic acid. The exopolysaccharides are a protectivesubstance forming a mucus which makes the bacteria particularly wellstorable on the seed/the plants.

[0022] In this context, the medium in which the bacteria are grownespecially preferably contains K₂HPO₄, KH₂PO₄, (NH₄)₂SO₄, MgSO₄ andsuccinic acid, preferable contents being approximately 6.0 g of K₂HPO₄,approximately 3.0 g of KH₂PO₄, approximately 1.0 g of (NH₄)₂SO₄,approximately 0.2 g of MgSO₄ and approximately 4.0 g of succinic acid inapproximately 1000 ml of deionized H₂O.

[0023] The inventor has found that exopolysaccharides are formedparticularly rapidly in this medium, the cell density in a solutionprepared by this method being less decisive for the seed treatment thanthe abovementioned conditioning.

[0024] On the other hand, the culture medium preferably containsglucose, preferred contents being approximately 6.0 g of K₂HPO₄,approximately 3.0 g of KH₂PO₄, approximately 1.5 g of (NH₄)₂SO₄,approximately 0.2 g of MgSO₄, approximately 2.0 g of glucose andapproximately 4 g of succinic acid in approximately 1000 ml of deionizedH₂O.

[0025] With this composition of the culture medium, a higher celldensity can be achieved since the concentration of the carbon sourcepresent is higher. However, the inventor has recognized that exclusivelyglucose in the culture medium would rapidly lead to an acidic pH, whichdoes not make possible further cultivation. This is why a mixture ofglucose and succinic acid is chosen as carbon source so that the glucoseis first metabolized, giving rise to a high cell density, but a low pH.After the glucose has been consumed, the succinic acid is metabolized,whereby the pH climbs, entailing survival, but slower growth, of thebacteria. This leads to the abovementioned formation ofexopolysaccharides and the simultaneous improved action ofmicroorganisms grown in such a solution.

[0026] Naturally, it is particularly preferred in this context to employan isolate of the species Proradix.

[0027] According to a further object, the invention relates to asolution prepared by the method described so far.

[0028] On the other hand, it is also possible to dry the solution invacuo, which is advantageous particularly when the culture mediumcontains glucose, so that the resultant powder contains bacteria in highconcentrations. This powder can then be dissolved in water for use inthe field, it being possible to employ the water treated thus forspraying seed, potatoes and the like.

[0029] According to yet another object, the present invention alsorelates to a powder prepared thus.

[0030] Furthermore, according to one object the invention relates to amethod for the treatment of seeds in which seed moistened with the novelsolution is temporarily exposed to sub-atmospheric pressure.

[0031] This is because the inventor of the present application hasrecognized that bacteria, in particular bacteria conditioned inaccordance with the novel method, preferably the species Proradix, aresuitable for the vacuum treatment of seed and lead to outstandingprotective results.

[0032] On the other hand, the invention also relates to a method inwhich the seed/the plants is/are sprayed with a solution of the novelpowder in water. This simple method, too, results in an effectiveprotection of the treated plants.

[0033] Finally, according to another object the invention also relatesto the use of the Pseudomonas strain Proradix for the treatment ofplants, and to plants and seed treated by the novel method or inaccordance with the novel use.

[0034] Further advantages will be seen from the following description ofexamples for growing and using the novel method and the speciesProradix.

[0035] Of course, the features which have been mentioned hereinabove andare yet to be demonstrated hereinbelow can be used not only in thecombinations stated specifically, but also in other combinations oralone, while still being within the scope of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS Example 1 Isolation of the SpeciesProradix

[0036] Soil samples and roots were suspended and plated in dilutionseries in a selective medium so that individual colonies where obtained.Fluorescent colonies were employed for an in vitro screening, in whichover 500 isolates were tested for inhibiting the growth ofphytopathogenic fungi, in particular the soil-dwelling fungusRhizoctonia. Candidates of interest were selected and tested on plantsand subsequently in field trials.

[0037] In these tests, the inventor of the present application hasisolated the species Proradix (DSM 13134) of the genus Pseudomonaswhich, according to identification by the DSMZ, is another specieswithin the RNA group I of the Pseudomonadaceae. A final attribution ofProradix was not possible, however, since only relatively littlesequence similarity exists with validly described species of the genusPseudomonas, but there is no doubt that the microorganism is afluorescent representative of the Pseudomonas RNA group 1.

Example 2 Culturing the Isolate

[0038] a) For the treatment of seeds, a Pseudomonas isolate, preferablythe species Proradix, is inoculated into the following culture medium:K₂HPO₄ 6.0 g KH₂PO₄ 3.0 g (NH₄)₂SO₄ 1.0 g MgSO₄ 0.2 g succinic acid 4.0g

[0039] The inoculated culture medium is incubated for 72 hours at 28°C./100 rpm; this gives a “liquid seed pickle”.

[0040] b) To prepare a pulverulent formulation, a Pseudomonas isolate,preferably the species Proradix, is grown in the following culturemedium: K₂HPO₄ 6.0 g KH₂PO₄ 3.0 g (NH₄)₂SO₄ 1.5 g MgSO₄ 0.2 g glucose2.0 g succinic acid 4.0 g

[0041] Depending on the concentration of the inoculum, the culturemedium inoculated thus is incubated for 65-70 hours at 28-30° C./100rpm.

[0042] The solution is subsequently made into a powder in a vacuumdrying oven. Formulation auxiliaries: dry skim milk and gum arabic.

[0043] c) Both culture media induce the formation of exopolysaccharides,while this formation takes place very rapidly in the case of the culturemedium described under a), the cell concentration is of no importance,since seed treatment does not require large amounts of bacteria.

[0044] However, a high cell concentration is indeed required in the caseof the medium described under b), while the formation ofexopolysaccharides is still desired. This is achieved by the mixture ofglucose and succinic acid. First, the glucose is metabolized, whichleads to a reduced pH, but to a high cell concentration. Once theglucose is consumed, the succinic acid is used as carbon source, and itsdegradation products lead to a rise in the pH and to the formation ofexopolysaccharides.

Example 3 Treatment of Seed with Proradix

[0045] One possible treatment consists in dissolving the powder preparedin Example 2b) in water and using it to spray the seed or the youngplants before planting.

[0046] Furthermore, it is also possible to spray the solution preparedwith the culture medium described in Example 2a) directly ontoseed/plants before planting.

[0047] If the treated seed is still to be stored before planting, it isfirst moistened with the solution described in Example 2a), and thenintroduced into a sealable chamber in which a temporary vacuum isgenerated.

[0048] In this manner, the bacteria penetrate the seed and can stillefficiently exert their protective action, even after prolonged storage.

Example 4 Treatment of Young Lettuce Plants of Variety Garunda

[0049] Young plants of variety Garunda which are to be planted in onehectare of arable land are treated by pouring a solution of 50 mlProradix (10⁷ cfu/ml) per plant (Example 2b) over the young plantsbefore they are planted up.

[0050] After harvesting, only 50% of the plants were not of marketablequality, while in the case of an untreated control 70% of the harvestedplants were not of marketable quality.

[0051] In a comparative treatment with the chemical fungicides Switsch(NOVARTIS) (0.8 kg/ha) and Risolex (Spiess Urania) (3 l/ha), 58 and 48%,respectively, were not of marketable quality.

[0052] This result demonstrates that the lettuce is markedly lesssusceptible to black rot following treatment with Proradix, and theresults are at least as good as in the case of treatment with chemicalfungicides.

Example 5 Treatment of Potatoes

[0053] Proradix is formulated as a powder as described in Example 2b),and the powder is dissolved in water as follows:

[0054] 60 g of powder per 80 l of water for 1 ha with seed potatoes

[0055] 60 g of powder

4×10¹² cfu

[0056] The potato tubers are sprayed automatically with this solutionand then planted directly. As an alternative, the potato tubers aredusted directly with the powder.

[0057] Comparative experiments between untreated potatoes (control)potatoes treated with Proradix and potatoes treated with FZB24 (BayerLeverkusen, base: Bacillus subtilis) gave approximately identical yieldsof marketable produce per hectare. However, the quality showed thattreatment with Proradix afforded reliable protection against black speck(Rhizoctonia attack), approximately 45% of the harvested potatoesshowing no symptoms and a further approximately 45% showing minorsymptoms. In the control group, the corresponding figures are 10% and50%, and 25% and 50% in the case of FZB24.

[0058] Again, superiority of Proradix to the conventional cropprotection products is demonstrated.

Example 6 Treatment of Carrots Sold Clean

[0059] Carrot seed is moistened with Proradix seed treatment of Example2a) as described in Example 3 and vacuum-treated (seed infiltration),stored and then sown.

[0060] The harvested carrots were stored for 4 months at 4° C., then—asis customary in sales—stored for two weeks at room temperature andfinally assessed.

[0061] The quality showed 50% without blemish for the variety Nerac(untreated control: 18%) and 27% of useless carrots (control: 58%). Thefigures for the variety Mocum were 36% (control: 14%) and 46% (control:54%), respectively.

[0062] Thus, the treatment of carrots sold clean with Proradix leads toa markedly increased product quality.

[0063] In conclusion, it can therefore be said that for the treatment ofplants and/or seed an isolate of useful bacteria, preferably of thegenus Pseudomonas, preferably the species Proradix, is provided andincubated in a culture medium containing phosphorus compounds, nitrogencompounds and succinic acid. The solution can be used directly forspraying plants and/or seed, optionally followed by vacuum treatment.Furthermore, the solution can be vacuum-dried, the powder beingdissolved in water prior to use.

What is claimed is:
 1. Pseudomonas strain termed Proradix, which wasdeposited under the provisions of the Budapest Treaty on Mar. 11, 1999at the DSMZ, 38124 Braunschweig, under the Accession number PRORADIX-DSM13134.
 2. A method for the preparation of a solution for the treatmentof plants or seed, comprising the steps of: a) providing an isolate ofthe genus Pseudomonas, b) providing a culture medium containingphosphorus compounds, nitrogen compounds and succinic acid, c)inoculating the culture medium with the isolate, and d) incubating theculture medium at approximately 26-32° C. with gentle shaking for atleast 50 hours.
 3. The method of claim 2, wherein the culture mediumcontains K₂HPO₄, KH₂PO₄, (NH₄)₂SO₄, MgSO₄ and succinic acid.
 4. Themethod of claim 3, wherein the culture medium contains glucose.
 5. Themethod of claim 3, wherein the culture medium contains approximately 6.0g of K₂HPO₄, approximately 3.0 g of KH₂PO₄, approximately 1.0 g of(NH₄)₂SO₄, approximately 0.2 g of MgSO₄ and approximately 4.0 g ofsuccinic acid in approximately 1000 ml of deionized H₂O.
 6. The methodof claim 4, wherein the culture medium contains approximately 6.0 g ofK₂HPO₄, approximately 3.0 g of KH₂PO₄, approximately 1.5 g of (NH₄)₂SO₄,approximately 0.2 g of MgSO₄, approximately 2.0 g of glucose andapproximately 4.0 g of succinic acid in approximately 1000 ml ofdeionized H₂O.
 7. The method of claim 2, wherein the bacterium isPseudomonas strain Proradix (DSM 13134).
 8. The method of claim 6,wherein, after incubation, the solution is vacuum-dried to prepare apowder.
 9. The method of claim 7, wherein, after incubation, thesolution is vacuum-dried to prepare a powder.
 10. The method of claim 5,wherein the bacterium is Pseudomonas strain Proradix (DSM 13134). 11.The method of claim 6, wherein the bacterium is Pseudomonas strainProradix (DSM 13134).
 12. A solution prepared by the method of claim 2.13. A solution prepared by the method of claim
 5. 14. A powder preparedby the method of claim
 8. 15. A powder prepared by the method of claim9.
 16. A method of treating seed or plants, comprising the steps of: a)moistening the seed or plants with the solution of claim 12, b) applyinga sub-atmospheric pressure to the seed or plants moistened above, and c)releasing the sub-atmospheric pressure.
 17. A method of treating seed orplants, comprising the steps of: a) moistening the seed or plants withthe solution of claim 13, b) applying a sub-atmospheric pressure to theseed or plants moistened above, and c) releasing the sub-atmosphericpressure.
 18. A method for treating seed or plants, comprising thesteps: a) dissolving the powder of claim 14 in water, and b) spraying orimmersion treatment of the seed or of seed potatoes with the solution ofstep a) or pouring the solution of step a) over young plants.
 19. Amethod for treating seed or plants, comprising the steps of: a)dissolving the powder of claim 15 in water, and b) spraying or immersiontreatment of the seed or of seed potatoes with the solution of step a)or pouring the solution of step a) over young plants.
 20. A method fortreating plants or seed comprising the step of treating the plants orseed with the Pseudomonas strain Proradix of claim
 1. 21. Seed or plantstreated by the method of claim
 16. 22. Seed or plants treated by themethod of claim
 17. 23. Seed or plants treated by the method of claim18.
 24. Seed or plants treated by the method of claim
 19. 25. Seed orplants treated by the method of claim 20.